Method for producing antibiotic C-15003 P-3

ABSTRACT

A novel Antibiotic C-15003 P-3 is specifically produced by cultivating a microorganism of the genus Nocardia in a culture medium containing valine or isobutyric acid and/or their derivatives. 
     The Antibiotic C-15003 P-3 is useful as an antifungal, antiprotozoan and antitumor agent.

This invention relates to a method for producing Antibiotic C-15003 P-3in an industrially advantageous method.

Antibiotic C-15003 P-3 [hereinafter, it is abbreviated as "P-3"] is anovel compound obtained by cultivating a microorganism of the genusNocardia, which is isolated from natural resources.

The microorganisms employable in the present invention, when cultured bythe use of conventional culture media, generally produce severalcomponents of Antibiotic C-15003 simultaneously. For separating eachcomponent from the cultured broth, very complicated processes arerequired, which inevitably invites a low yield of the component desired.

With the purpose of overcoming this drawback, the present inventors havemade extensive study, especially, for recovering P-3 specifically, andfound that P-3 can be produced with a remarkably high ratio to the totalcontent of Antibiotic C-15003, when the culture medium of a specificcomposition is employed.

The present invention is a method for producing Antibiotic C-15003 P-3characterized in that said method comprises cultivating a microorganismbelonging to the genus Nocardia and capable of producing AntibioticC-15003 P-3 [hereinafter sometimes called "Antibiotic C-15003P-3-producing strain] in a culture medium containing valine orisobutyric acid or their derivatives or salts thereof, to specificallyproduce Antibiotic C-15003 P-3 in the cultured broth, and recoveringAntibiotic C-15003 P-3 from the broth.

In the context of this invention, the term "Antibiotic C-15003" or"C-15003" means, generically, the four compounds having the followinggeneral formula (I) as a group, or a mixture of two or three of saidcompounds or, severally, any one of the same compounds. ##STR1##

Referring, also, to the general formula (I), the compound in which R is--CO--CH₂ --CH₃ is referred to herein as "Antibiotic C-15003 P-2" ormore briefly as "P-2"; the compound in which R is ##STR2## is referredto herein as "Antibiotic C-15003 P-3" or more briefly as "P-3"; thecompound in which R is --CO--CH₂ --CH₂ --CH₃ is referred to herein as"Antibiotic C-15003 P-3'", or, more briefly, as "P-3'"; the compound inwhich R is ##STR3## is referred to herein as "Antibiotic C-15003 P-4"or, more briefly, as "P-4".

As an example of the Antibiotic C-15003 P-3-producing strain ofmicroorganism, there may be mentioned an actinomycete Strain No. C-15003(hereinafter sometimes abbreviated as "Strain No. C-15003") which wasisolated from soil and other samples in the screening forantibiotic-producing microorganisms.

The microbiological characters of Strain No. C-15003 were investigatedby procedures analogous to those proposed by Schirling & Gottlieb[International Journal of Systematic Bacteriology 16, 313-340 (1966)].The results of observations at 28° C. over 21 days are as follows.

(1) Morphological characters

The vegetative mycelium extends well and develops into branches, both onagar and in liquid medium. Many of the hyphae measure 0.8 to 1.2 μm indiameter and, in certain instances, may divide into fragments resemblingrod bacteria or branched hyphae of short length. The strain gives goodgrowth on various taxonomical media, with aerial mycelium beingsuperimposed on the vegetative mycelium, although it frequently formscoremia like bodies (50-200×200-1000 μm) on which further aerial growthtakes place. Many of the aerial mycelia are flexuous, straight or aloosely spiral like configuration being encountered on a few occasions.Microscopic examination of aged cultures reveals that only in few casesthe conidia like cells occur in chains, while the cell suspensionsobtained from the surfaces of such cultures, as microscopicallyexamined, contained many elongated ellipsoidal (0.8-1.2 μm×4.8-6.8 μm)and ellipsoidal (0.8-1.2×1.0-2.0 μm) bodies resembling arthrospores.

Electron-microscopic examinations showed that these bodies had smoothsurfaces.

(2) The constituents of cells

The strain was shake-cultured in modified ISP No. 1 medium at 28° C. for66 to 90 hours, at the end of which time the cells were collected andrinsed. By the method of B. Becker et al. [Applied Microbiology 12, 421(1964)] and the method of M. P. Lechevalier [Journal of Laboratory andClinical Medicine 71, 934 (1968)], the above whole cells were examinedfor diaminopimelic acid and sugar composition. The former was found tobe the meso-form, while spots were detected which corresponded togalactose and arabinose.

(3) Characteristics on taxonomical media

The strain showed comparatively good growth on various media, with thevegetative mycelium being colorless to pale yellow in initial phases ofculture and light yellowish tan to yellowish tan in later phases. Thestrain produces soluble pigments, yellow to yellowish tan, in varioustaxonomical media. The aerial mycelium is powdery and generally givesmoderate growth, being white to yellow or light yellowish tan. Thecharacteristics of the strain in various taxonomical media are set forthin Table 1.

TABLE 1 Cultural characteristics of Strain No. C-15003 on taxonomicalmedia

(A) Sucrose nitrate agar:

Growth (G): Moderate, Brite Melon Yellow (3 ia)* to Amber tan (3 lc)*,coremia like bodies formed

Aerial mycelium (AM): Scant, white

Soluble pigment (SP): None or pale yellowish tan

(B) Glycerol nitrate agar:

G: Moderate, Lt Ivory (2 ca)*, coremia like bodies formed

AM: Moderate, white

SP: None

(C) Glucose asparagine agar:

G: Moderate, Brite Marigold (3 pa)* to Brite Yellow (2 pa)*.

AM: Scant, white

SP: Brite Yellow (2 pa)*

(D) Glycerol asparagine agar:

G: Moderate, Lt Ivory (2 ca)*, coremia like bodies formed

AM: Scant, white

SP: None

(E) Starch agar:

G: Moderate, Lt Ivory (2 ca)* to Lt Wheat (2 ea)*, coremia like bodiesformed

AM: Abundant, Lt Ivory (2 ca)*

SP: None

(F) Nutrient agar:

G: Moderate, Lt Ivory (2 ca)* to Colonial Yellow (2 ga)*, coremia likebodies formed

AM: Scant, white

SP: None

(G) Calcium malate agar:

G: Moderate Lt Ivory (2 ca)* to Lt Wheat (2 ea)*, coremia like bodiesformed.

AM: Moderate, white to Lt Ivory (2 ca)*

SP: None

(H) Yeast extract-malt extract agar:

G: Moderate, Amber (3 lc)* to Brite Yellow (3 la)*, coremia like bodiesformed

AM: Moderate, white to Lt Ivory (2 ca)*

SP: None

(I) Oatmeal agar:

G: Moderate, Lt Ivory (2 ca)* to Colonial Yellow (2 ga)*, coremia likebodies formed

AM: Scant, white to light yellow

SP: None

(J) Peptone yeast extract iron agar:

G: Moderate, Colonial Yellow (2 ga)*

AM: None

SP: Colonial Yellow (2 ga)*

(K) Tyrosine agar

G: Moderate, Lt Ivory (2 ca)* to Lt Melon Yellow (3 ea)*, coremia likebodies formed.

AM: Moderate, white to Lt Ivory (2 ca)*,

SP: Camel (3 ie)*

(4) Physiological characters

The physiological characters of the strain are shown in Table 2.Temperatures range for growth: 12° C. to 38° C. The temperature range inwhich good aerial growth occurs on agar (ISP No. 2) is 20° to 35° C.

TABLE 2 The physiological characters of Strain No. C-15003.

Temperature range for growth: 12° to 38° C.

Temperature range for aerial growth: 20° to 35° C.

Liquefaction of gelatin: Positive

Hydrolysis of starch: Positive

Reduction of nitrates: Positive

Peptonization of milk: Positive

Coagulation of milk; Negative

Decomposition of casein: Positive

Production of melanoid pigments:

Negative (peptone yeast extract iron agar),

Positive (tyrosine agar)

Lecomposition of tyrosine: Positive

Decomposition of xanthine: Negative

Decomposition of hypoxanthine: Negative

Tolerance to lysozyme: Positive

Tolerance to sodium chloride: 2%

(5) Utilization of various carbon sources

The utilization of various carbon sources was investigated using amedium described in Pridham and Gottlieb [Journal of Bacteriology 56,107 (1948)] and a basal medium of the same composition plus 0.1% ofyeast extract. The resultant spectrum is shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        The utilization of carbon sources by Strain                                   No. C-15003                                                                   Source of carbon                                                                         Growth     Sources of carbon                                                                          Growth                                     ______________________________________                                        D-Xylose   +       ++*    Raffinose  ± ±*                               L-Arabinose                                                                              +       +      Melibiose  +    +                                   D-Glucose  ++      ++     i-Inositol -    -                                   D-Galactose                                                                              +       +      D-Sorbitol -    -                                   D-Fructose +++     ++     D-Mannitol ++   ++                                  L-Rhamnose +       +      Glycerol   -    ±                                D-Mannose  +++     ++     Soluble starch                                                                           +    +                                   Sucrose    ++      ++     Control (None)                                                                           -    -                                   Lactose    -       -                                                          Maltose    ±    +                                                          Trehalose  +       ++                                                         ______________________________________                                         *Basal medium with 0.1% yeast extract added                                   Note:                                                                         +++: Luxuriant growth                                                         ++: Good growth                                                               +: Growth                                                                     ±: Poor growth                                                             -: No growth                                                             

(6) Other characteristics

The cells were harvested by the procedure previously described in (2)and DNA was prepared by a procedure analogous to that of J. Marmur etal. [Journal of Molecular Biology 3, 208, 1961]. The G-C(guanine-cytosine) content of the DNA was found to be about 71 mole %.

Gram-staining of the vegetative mycelium of this strain was positive.

The above characteristics of Strain No. C-15003 were compared with thedescriptions in S. A. Waksman's "The Actinomycetes Vol. 2" [The Williamsand Wilkins Co., 1961]; R. E. Buchanan and N. E. Gibbons, "Bergey'sManual of Determinative Bacteriology, 8th Ed., 1974"; and otherliteratures.

Whilst this strain was thought to belong to Group III of the genusNocardia, the failure to find any species having the characters so fardescribed among the known species led us to conclude that this strainrepresented a novel species of microorganism.

The present Strain No. C-15003 has been deposited at FermentationResearch Institute, Agency of Industrial Science and Technology (FERM),Japan under the deposition number of FERM-P No. 3992; at The Institutefor Fermentation, Osaka (IFO), Japan under the accession number of IFO13726 and at The American Type Culture Collection (ATCC), Maryland,U.S.A. under the accession number of ATCC-31281.

While a microorganism of the genus Nocardia is liable, as aremicroorganisms generally, to undergo variations and mutations, eitherspontaneously or artificially. For example, the many variants of thestrain which are obtainable by irradiation with X-ray, gamma rays,ultraviolet light, etc., by single cell isolation, by culture on mediacontaining various chemicals, or by any other mutagenic treatment, aswell as the mutants spontaneously derived from the strain, should not beconsidered to represent any other distinct species but, rather, any ofsuch variants and mutants capable of producing C-15003 P-2, P-3, P-3'and/or P-4 may be invariably utilized for the purposes of thisinvention. By way of example, subjecting Strain No. C-15003 to variousmutagenic treatments yields mutants substantially lacking the ability toproduce soluble pigments, mutants with substrate mycelia which arecolorless, yellowish green, reddish tan or orange-red, mutants whosehyphae are ready to fragment into bacillary elements or branched shorthyphal fragments, and mutants with abundant white aerial mycelia orsubstantially without aerial mycelia.

Valine and/or isobutyric acid, as additive substances in the presentinvention, may be used as forms of derivatives. Examples of thederivatives are esters such as alkyl esters having one to 2 carbon atoms(e.g. methyl ester, ethyl ester) of the above compounds, amides such asamide or alkyl amide having one to 2 carbon atoms (e.g. N-methyl amide,N-ethyl amide) of the above compounds, its keto-acid (e.g.α-ketoisovaleric acid), salts of the above compounds such ashydrochloride, sodium salt, potassium salt or calcium salt. Valine maybe used as its D-form, L-form or DL-form. The above additive substancesmay be mixtures of valine, isobutyric acid and/or their derivatives.

In the practice of this invention, the aforesaid substances aregenerally added to the medium about 0.01 to 1.0% (W/V), preferably about0.1 to 0.5% (W/V), at any time of the cultivation as long as thecultivation of Nocardia sp. No. C-15003 are carried out, preferably atthe initial stage of the cultivation.

The medium employed for the cultivation of such an Antibiotic C-15003P-3-producing strain may be whichever of a liquid and a solid mediumonly if it contains nutrients which the strain may utilize. Forhigh-production runs a liquid medium is generally preferred. The mediummay comprise the additive substance used in the present invention,carbon and nitrogen sources which Strain No. C-15003 may assimilate anddigest, inorganic matter, trace nutrients, etc. As examples of saidcarbon sources may be mentioned glucose, lactose, sucrose, maltose,dextrin, starch, glycerol, mannitol, sorbitol, etc., fats and oils (e.g.soybean oil, lard oil, chicken oil, etc.) and so forth. The nitrogensources may for example be meat extract, yeast extract, dried yeast,soybean meal, corn steep liquor, peptone, cottonseed flour, molasses,urea, ammonium salts (e.g. ammonium sulfate, ammonium chloride, ammoniumnitrate, etc.), nitrate salts (e.g. sodium nitrate, potassium nitrate).The medium may further contain salts of sodium, potassium, calcium,magnesium, iron, manganese, zinc, cobalt, nickel, etc. salts ofphosphoric acid, boric acid, etc. Further, the medium may contain, asadded, vitamins (e.g. B₁, B₂, nicotinic acid, B₁₂, C, E, etc.), nucleicacids (e.g. purine, pyrimidine and derivatives thereof) and so forth.For the purpose of adjusting the pH of the medium, a mineral acid and/oran alkaline metal or ammonia as well as the corresponding bases as thepH-adjusting agents may be added. Furthermore oils and fats,surfactants, and antifoaming agents may also be added into the medium,if desired.

The cultivation may be conducted by any of the stationary, shaking,aerobic submerged and other cultural conditions. For high productionruns aerobic submerged culture is of course preferred. While theconditions of culture, of course, depend upon the condition of thefermentation and the composition of the medium, the strain used,cultural method and other factors, it is normally preferred to carry outincubation at 20° to 35° C. with an initial pH of about 5.5-8.5 orthereabouts, particularly from 23° to 30° C. with an initial pH of 6.5to 7.5. While the cultivation time is also variable according to thesame factors as mentioned above, it is advisable to continue thecultivation until the potency of P-3, becomes maximal. In the case ofshake culture or aerobic submerged culture in liquid medium, the timerequired normally ranges from about 48 to 240 hours.

The following is a concrete example of the method for the production ofP-3. Strain No. C-15003 was inoculated into a culture medium (I)composing 3% soluble starch, 0.2% ammonium chloride, 0.05% magnesiumsulfate, 1.09% potassium dihydrogenphosphate, 2.09% dipotassiumhydrogen-phosphate, 0.001% ferrous sulfate and the additive substance,or a culture medium (II) composing 5% dextrin, 3% corn steep liquor,0.1% peptone, 0.5% calcium carbonate and the additive substance, andcultivated at 28° C. for 144 hours on a rotary shaker (200 r.p.m.) orfermentor.

Tables 4 and 5 show the results using the media (I) and (II),respectively.

The potency of the C-15003 was assayed by paper disc method withTalaromyces avellaneus IFO 7721 as an assay organism. The assay mediumwas consisting of 3.5 g disodium hydrogenphosphate, 0.5 g potassiumdihydrogenphosphate, 5 g yeast extract (Difco U.S.A.), 10 g glucose, 15g of agar and 1000 ml distilled water (pH 7.0). The determination ofP-2, P-3 and P-4 accumulated in the cultured broth was conducted asfollows:

The cultured broth was extracted with the equal volume of ethylacetate.The solvent layer was concentrated and dried. The dried matter wasdissolved with ethylacetate to give 1/100-volume of the starting broth.The products were developed on a silica-gel thin-layer chromatography(silica-gel plate 60F₂₅₄, Merck, West Germany) with water-saturatedethylacetate. The amount of the antibiotic and the ratio of thecomponents were determined by Shimazu Dual-wave length TLC-scanner modelCS-910 (Shimazu, Ltd., Japan) on the basis of the integral-densities ofthe each spot on the chromatogram at 254 nm. Ratio of the components wasrepresented as weight-by-weight percent in the total products, P-2, P3and P-4.

As shown in Tables 4 and 5, Strain No. C-15003 produced the antibioticP-3 in the ratio of 90% or more of the total in the presence of thespecific additives, but 65% or less in the absence of such thecompounds. Therefore, the recovery of P-3 from the cultured brothbecomes more efficiently in the former case than that in the latter.

                                      TABLE 4                                     __________________________________________________________________________          Amount     Ratio of                                                           of   Time of                                                                            components                                                                             Total                                                Additive                                                                            addition                                                                           addition                                                                           (W/W %)  potency                                              substance                                                                           (%)  (hour)*                                                                            P-2                                                                              P-3                                                                              P-4                                                                              (μg/ml)                                           __________________________________________________________________________    None  --   --   10 65 25 10                                                   L-valine                                                                            0.1  0    1  95 4  16                                                   L-valine                                                                            0.3  0    1  97 2  26                                                   L-valine                                                                            0.1  72   2  95 3  10                                                   D-valine                                                                            0.1  0    2  95 3  16                                                   sodium                                                                        isobutyrate                                                                          0.01                                                                              0    1  91 8   8                                                   __________________________________________________________________________     *Zero time means that the additive substance was added into the medium as     one of the ingredients.                                                  

                  TABLE 5                                                         ______________________________________                                                Amount            Ratio of                                                    of       Time of  components Total                                    Additive                                                                              addition addition (W/W %)    potency                                  substance                                                                             (%)      (hour)*  P-2  P-3  P-4  (μg/ml)                           ______________________________________                                        None    --       --       15   60   25   18                                   L-valine                                                                              0.1      0        5    90   5    15                                   L-valine                                                                              0.3      0        3    94   3    15                                   L-valine                                                                              0.5      0        2    96   2    13                                   L-valine                                                                              0.3      48       3    94   3    13.5                                 sodium                                                                        isobutyrate                                                                           0.3      0        5    92   3    11                                   ______________________________________                                         Note:                                                                         Zero time has the same meaning as the Note of Table 4.                   

Because P-3, which is thus produced in the cultured broth, is lipophylneutral substance, it can be conveniently recovered from the culturedbroth by separation and purification procedures which are ordinallyemployed for the recovery of such microbial metabolites. P-3 is easilyextracted from the culture filtrate into water-immiscible organicsolvents such as fatty acid esters, e.g. ethyl acetate and amyl acetate;alcohols, e.g. butanol; halogenated hydrocarbons, e.g. dichloromethane,chloroform; and ketones, e.g. methyl isobutyl ketone. The extraction ofP-3 from the filtrate is carried out at a pH near neutral, preferablywith ethyl acetate at pH 7. The extract is washed with water andconcentrated under reduced pressure. Then, a nonpolar solvent such aspetroleum ether or hexane is added to the concentrate and the crudeproduct containing the active compounds is recovered as precipitates.The crude product is sequentially subjected to convenient purificationprocedures, if desired. Thus, as a routine purification procedure,adsorption chromatography is useful and, for this purpose, one of thosecommon adsorbents such as silica gel, active alumina,macroporous-nonionic adsorbent resin, etc., may be employed. P-3 in thecrude product is developed on such the silica-gel chromatography with,for example, petroleum ether and hexane and eluted by the addition of apolar solvent such as ethyl acetate, acetone, ethanol or methanol, or ahalogenated hydrocarbon such as dichloromethane or chloroform,containing a polar solvent such as an alcohol, e.g. methanol or ethanol,a ketone, e.g. acetone or methyl ethyl ketone, or the like. In this way,P-3 is eluted separated and recovered.

In the case that a macroporous adsorbent resin is used for thepurification of P-3, elution of P-3 from the column is accomplished witha mixture of water with a lower alcohol, a lower ketone or an ester. Thelower alcohol may, for example, be methanol, ethanol, propanol orbutanol, etc., and the lower ketone may for example be acetone or methylethyl ketone, etc. The ester may for example be ethyl acetatebutylacetate, etc. In a typical procedure, the crude product isdissolved in 60% methanol-water and adsorbed on a column of Diaion HP-10(Mitsubishi Chemical Industries, Ltd., Japan). The column is washed with70% methanol-water and P-3 is eluted with 90% methanol-water.

In the process described above, the fractions containing P-3 are pooledand concentrated under reduced pressure. To the dry product is added 5to 8 volumes of ethyl acetate and the mixture is allowed to stand,whereupon P-3 is crystallized out.

In the method of the present invention, the production ratio of P-3 inthe C-15003 products reaches 90% or more and P-3 is easily recoveredfrom the cultured broth. Therefore, the method of the present inventionis very advantageous for an industrial production of P-3.

The physico-chemical properties of P-3 obtained in Example 4 are shownin Table 6.

                  TABLE 6                                                         ______________________________________                                        Antibiotic C-15003 P-3                                                        ______________________________________                                                        C.sub.32 H.sub.43 ClN.sub.2 O.sub.9 = 635.169                 Melting point (°C.)                                                                    190°-192°                                       Specific rotation                                                                             -136° ± 10°                                  (α).sub.D.sup.22°                                                                (C = 0.375 CHCl.sub.3)                                        Elemental analysis                                                                            C           60.06                                                             H           7.04                                              Found (%)                                                                                     N           4.33                                                              Cl          5.37                                              Elemental analysis                                                                            C           60.51                                                             H           6.82                                              Calcd. (%)                                                                                    N           4.41                                                              Cl          5.58                                              Ultraviolet     233(30250) 240(sh 28450)                                      absorption spectra                                                                            252(27640) 280(5750)                                          nm(ε)   288(5700)                                                     (in methanol)                                                                 Infrared        1740, 1730, 1670, 1580                                        absorption spectra                                                                            1445, 1385, 1340, 1255                                        (cm.sup.-1)KBr  1180, 1150, 1100, 1080,                                                       1038                                                          Nuclear magnetic                                                                              1.27(d) (3H)                                                  resonance spectra                                                                             1.28(d) (3H)                                                  (ppm)                                                                         100MHz in CDCl.sub.3                                                          Mass spectra(m/e)                                                                             573, 485, 470, 450                                            Solubility      Insoluble in petr.ether,                                                      hexane, water.                                                                Sparingly soluble in                                                          benzene, ether.                                                               Soluble in chloroform,                                                        ethyl acetate, acetone,                                                       ethanol, methanol,                                                            pyridine, tetrahydrofuran,                                                    dimethylsulfoxide.                                            Color reactions Dragendorff: Positive                                                         Beilstein: Positive                                           ______________________________________                                    

It is assumed that P-3, P-3' and P-4 are novel compounds, but P-2 is thesame compound as maytansinol propionate which is shown in Kupchan etal's report [The Journal of American Chemical Society 97, 5294 (1975)]in terms of elemental analysis, specific rotation, ultraviolet rayabsorption, infra red ray absorption, mass spectrum and so forth.

Biological activity of P-3 is as follows:

(A) Antimicrobial activity:

With trypticase-soy agar (BBL) as an assay medium, the inhibitoryconcentrations against the microorganisms described below wereinvestigated by the paper disc method. Filter-paper discs (ToyoSeisakusho, thin-type, 8 mm in dia.) each impregnated with 0.02 ml of a300 μg/ml solution of P-3 were placed on agar plates respectivelyinoculated with the microorganisms described below. P-3 had no activityagainst the following bacteria: Escherichia coli, Proteus vulgaris,Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus,Bacillus subtilis, Bacillus cereus, Klebsiella pneumoniae, Serratiamarcescens, and Mycobacterium avium.

On the other hand, the growth of a fungus, Talaromyces avellaneus isinhibited by P-3 on an agar plate consisting of 3.5 g disodium hydrogenphosphate, 0.5 g monopotassium dihydrogen phosphate, 5 g yeast extract(Difco), 10 g glucose, 15 g agar, 1000 ml distilled water, pH 7.0. Theminimal inhibitory concentration was 3 μg/ml for P-3.

Furthermore, Tetrahymena pyriformis W as an assay organism wascultivated on an assay medium [composed of 20 g Proteose-peptone(Difco), 1 g yeast extract, 2 g glucose, 1000 ml distilled water and 10ml 1 M-phosphate buffer, pH 7.0,] at 28° C. for 44 to 48 hours and thegrowth inhibitory activity of P-3 against the protozoa was determined byserial dilution method. Growth inhibition occurred at 1 μg/ml.

P-3 had the activity against the following microorganisms:

Fusicladium levieri, Helminthosporium sigmoidium var irregulare,Pyricularia oryzae, Cochlioborus miyabeanus, Sclerotinia screrotiorum,Pellicularia sasakii, Trichophyton rubrum, Rhodotorula rubra andCryptococcus neoformans.

(B) Antitumour activity

The therapeutic effects of P-3 (dosed intraperito-nearly for 9consecutive days) upon P388 leukemia in mice (1×10⁶ cells/animal, mouse,intra-peritoneally transplanted) was investigated. P-3 had an antitumouractivity as high as 168% life-span-extending ratio at the dose level of0.00625 mg/kg/day.

(C) Toxicity

In a preliminary acute toxicity test with mice as test animals, whichinvolved intraperitoneal injection of P-3, this antibiotic showed a LD₅₀value more than 0.313 mg/kg.

As mentioned hereinbefore, P-3 has strong inhibitory activity againstfungi and protozoa and, therefore, is of value as an antifungal orantiprotozoan agent. Furthermore, because P-3 displays a lifespan-extending action upon tumour-bearing mammalian animals (e.g.mouse), it is also expected that the compound will be of use as anantitumour drug.

P-3, as an antifungal and antiprotozoan agent, can be used withadvantage for an assessment of the bacterial ecology in soil, activesludge, animal body fluid or the like. Thus, when valuable bacteria areto be isolated from soil samples or when the actions of bacteria are tobe evaluated independently of those of fungi and protozoa in connectionwith the operation and analysis of an active sludge system used in thetreatment of waste water, as the present antibiotic may be utilized toobtain a selective growth of the bacterial flora without permittinggrowth of the concomitant fungi and protozoa in the specimen. In atypical instance, the sample is added to a liquid or solid medium and0.1 ml of a 10 to 100 μg/ml solution of P-3 in 1% methanol-water isadded per ml of the medium, which is then incubated.

P-3 can also be used as an anti-microbial agent for the treatment ofplant diseases caused by the microorganisms mentioned in the above. Inthe typical application, P-3 is used in a form of 1% methanolic aqueoussolution containing 0.5 μg/ml-5 μg/ml of the antibiotic. For instanceP-3 may be used for the control of the blast, the Helminthosporium leafspot and the sheath blight of rice plants.

The following examples are further illustrative to explain the presentinvention in detail, wherein "part(s)" is based on weight unlessotherwise noted and the relationship between "part(s)" and "part(s) byvolume" corresponds to that between "gram(s)" and "milliliter(s)", and"%" is based on "weight/volume" unless otherwise noted.

EXAMPLE 1

Forty parts by volume of seed culture medium (1.0% glucose, 2.0%Bactotryptone and 1.2% Bacto-yeast extract, pH 7.0) is poured into 200parts by volume of Erlenmeyer flask.

After sterilization, Nocardia sp. No. C-15003 (IFO 13726; ATCC 31281;FERM-P No. 3992) was inoculated to the medium. The inoculant wasincubated at 28° C. on a rotary shaker (200 r.p.m.) to give a seedculture.

The cells in culture was washed three times with sterilized distilledwater, and the washed cells were reslurried in the original broth volumeof sterilized distilled water. One part by volume of the above wasinoculated into 40 parts by volume of main culture medium consisting of3% soluble starch, 0.2% ammonium chloride, 0.05% magnesium sulfate,1.09% potassium dihydrogenphosphate, 2.09% dipotassiumhydrogenphosphate, 0.001% ferrous sulfate and 0.1% L-valine and the maincultivation was conducted at 28° C. for 8 days on the rotary shaker (200r.p.m.).

The total production amount of C-15003 was 16 μg/ml, and the ratio ofP-3 in the total was 95% (W/W).

EXAMPLE 2

500 parts by volume of the seed culture as shown in Example 1 wasinoculated into 2,000 parts by volume of Sakaguchi flask and cultivatedat 28° C. for 48 hours on a reciprocal shaker (110 strokes/min.) to givean inoculum. The inoculum was transferred to 100×10³ parts by volume ofa medium consisting of 2.0% glucose, 3.0% soluble starch, 1.0% cornsteep liquor, 1.0% soybean flour, 0.5% polypepton (Daigo NutritiveChemicals, Ltd., Japan.), 0.3% sodium chloride and 0.5% calciumcarbonate, pH 7.0, in 200×10³ parts by volume of a stainless steelfermentor.

The cultivation was conducted at 28° C., for 48 hours under 100×10³parts by volume/minute aeration and 200 r.p.m. agitation. The culturebroth (10×10³ parts by volume) was transferred to 100×10³ parts byvolume of a fermentation medium (5% dextrin, 3% corn steep liquor, 0.1%peptone, 0.3% L-valine and 0.5% calcium carbonate, (W/V), pH 7.0) was in200×10³ parts by volume of the stainless steel fermentor. Thecultivation was carried out at 28° C. for 4 days under 100×10³ parts byvolume/minute aeration, 150 r.p.m. agitation.

The total production amount of C-15003 was 12 μg/ml, and P-3 in C-15003was about 98% (W/W).

EXAMPLE 3

To 95×10³ parts by volume of the cultured broth obtained in Example 2 isadded 50×10³ parts by volume of acetone. The mixture is stirred for 30minutes. To the resultant is added 2×10³ parts of Hyflo Super-Cel(Johnes and Manville Products, Ltd.) and the mixture is stirred well.The mixture was filtered with a pressure filter to give 135×10³ parts byvolume of filtrate. To the filtrate was added 50×10³ parts by volume ofwater and 90×10³ parts by volume of ethyl acetate, and the mixture wasstirred and extracted twice. The obtained ethyl acetate layer werecombined and washed twice with water of 80×10³ parts by volume each. Tothe ethylacetate layer obtained was added 1×10³ parts of anhydroussodium sulfate, dried and concentrated to 200 parts by volume. To theconcentrate was added petroleum ether, and emerged precipitate wasrecovered by filtration to give 35 parts of the crude product.

To thus obtained crude product was added 50 parts by volume of ethylacetate and the mixture was stirred. The insoluble were removed byfiltration and to the filtrate is added 10 parts of silica gel (Merck,West Germany, 0.05 to 0.2 mm). After stirring the mixture, ethyl acetatewas removed by distillation under reduced pressure.

The residue was applied to the top of a silica-gel column (500 parts byvolume). The antibiotics was eluted stepwise with 500 parts by volume ofn-hexane, 500 parts by volume of a mixture of n-hexane-ethyl acetate(3:1), 2,000 parts by volume of a mixture of n-hexane-ethyl acetate(1:1), and 2,000 parts by volume of water-saturated ethyl acetate, andthe eluates were collected in 50 parts by volume fractions, each.

One part by volume portion of each fraction was concentrated to dryness,and 0.1 part by volume of ethyl acetate was added to the concentrate togive a mixture. The mixture was spotted at 2.5 cm from the bottom edgeof a silica gel-glass plate (Merck, West Germany, 60 F₂₅₄, 0.25 mm,20×20) and developed for about 17 cm with water-saturated ethyl acetate.After development, detection was carried out with ultraviolet light(2537 A). The active fractions of Rf 0.42 were collected andconcentrated under reduced pressure to about 2 parts by volume. To thisconcentrate was added 20 parts by volume of petroleum ether to obtain9.1 parts of a crude crystals. The crude crystals was dissolved in 20parts by volume of warm ethyl acetate. After cooling, 0.85 part of P-3crystals were recovered. The purity of P-3 crystals obtained was 97%(W/W), and its melting point was 189° to 190° C.

EXAMPLE 4

In 400 parts by volume of 50% methanol was dissolved 20 parts of thecrude product obtained in Example 3. 1,000 parts by volume of DiaionHP-10 (Mitsubishi Chemical Industries Ltd., Japan) was packed into acolumn (2.5 cm in dia.) with 3,000 parts by volume of 50%methanol-water. The sample solution prepared above was passed throughthe column and washed with 1000 parts by volume of 60% methanol, andgradient elution was carried out continuously using 7,500 parts byvolume of 60% methanol-water and 7,500 parts by volume of 95%methanol-water.

The eluate was collected in 75 parts by volume fractions and eachfraction was applied on the silica-gel thin-layer chromatography,described in Example 3.

The active fractions Nos. 145 to 153 were collected and concentrated. Tothe concentrate were added 500 parts by volume of water and 1,000 partsby volume of ethyl acetate.

The mixture was shaken in a separatory funnel and the water layer wasseparated, and after washing twice with 300 parts by volume of water,the ethyl acetate layer was dried over anhydrous sodium sulfate,concentrated and allowed to stand.

The resulting crystals of P-3 were collected by filtration and dried(0.880 part of P-3). The purity of P-3 crystals obtained was 95% W/W andits melting point was 188° to 190° C.

EXAMPLE 5

Methyl ester of valine, N-methyl ester of valine, hydrochloride ofvaline, α-ketoisovaleric acid, methyl ester of isobutyric acid, N-methylester of isobutyric acid or a mixture of valine and isobutyric acid isused in place of L-valine in Example 1, whereby similar results, i.e.specific production of the objective P-3, are obtained.

What we claim is:
 1. In a method for producing Antibiotic C-15003 P-3 bycultivating a microorganism belonging to the genus Nocardia and beingcapable of producing Antibiotic C-15003 P-3 in a culture mediumcontaining assimilable carbon sources, and digestible nitrogen sources,wherein the improvement comprises incorporating about 0.01 to 1% byweight based on the volume of the culture medium of valine, isobutyricacid, α-ketoisovaleric acid or its salt or ester, amide or salt ofvaline or isobutyric acid as additive substances into the culturemedium.
 2. A method as claimed in claim 1, wherein the microorganism isNocardia sp. No. C-15003 (ATCC-31281; IFO 13726; FERM-P No. 3992).
 3. Amethod as claimed in claim 1, wherein the additive substance comprisesvaline and/or isobutyric acid.